The spread of antibiotic resistant bacteria and the possibility that antibiotic resistant strains could be used as agents of bioterrorism poses a significant health risk to the U.S. population and has necessitated the development of new classes of antibiotics. Among the enzymes that have attracted attention recently as potential antibiotic targets are the aminoacyl-tRNA synthetases. Recent evidence that differences exist between the catalytic mechanisms of the bacterial and human tyrosyl-tRNA synthetases, as well as the isolation of a compound that selectively inhibits bacterial tyrosyl-tRNA synthetases, suggests that this enzyme is a potential target for antibiotics. The long-term goal of this proposal is to understand how the aminoacyl-tRNA synthetases catalyze the aminoacylation of their cognate tRNAs. In particular, this proposal addresses the question of how tyrosyl-tRNA synthetase specifically recognizes tRNA (Tyr). There are three specific aims: (1) Verify that the recently solved crystal structure of the Thermus thermophilus tyrosyl-tRNA synthetase-tRNA (Tyr) complex is representative of the structure in solution, (2) Elucidate the physical basis for discrimination between cognate and non-cognate tRNAs by tyrosyl-tRNA synthetase, and (3) Determine how the Tyr-tRNA (Tyr) product is released from tyrosyl-tRNA synthetase. Pre-steady state kinetics, surface plasmon resonance, and isothermal titration calorimetry will be combined with site-directed mutagenesis to investigate the roles that conserved amino acids play in the recognition of tRNA by tyrosyl-tRNA synthetase. In addition to being relevant to developing tyrosyl-tRNA synthetase as an antibiotic target, this proposal is also relevant to the design of orthogonal aminoacyl-tRNA synthetase tRNA pairs that can be used to incorporate unnatural amino acids into proteins. The investigations described in this proposal will also complete the energetic analysis of the tyrosyl-tRNA synthetase reaction cycle. This will make tyrosyl-tRNA synthetase the first aminoacyl-tRNA synthetase in which both the energetic and structural changes have been elucidated for the entire reaction pathway.